The overall objective of this project is to gain deeper understanding of the mechanism controlling Ca/2+ release from the sarcoplasmic reticulum (SR) in skeletal muscle. Depolarization of the transverse tubules (TT) in a muscle fiber activates SR Ca/2+ release channels at triad junctions. We have recently used the Ca/2+ indicator dye fluo-3 with laser scanning confocal microscopy to detect discrete SR Ca/2+ release events ("Ca/2+ sparks"), which may arise from the opening of a few SR Ca/2+ channels or perhaps even from a single channel in functioning muscle fibers. These events, which originate at triad junctions and can be discerned at low levels of SR release activation, can be gated either by fiber depolarization or by physiological cytosolic ligands. They thus offer a unique window into the operation of SR Ca/2+ release channels within the normal structural and molecular environment in the muscle. We will characterize the frequency and pattern of occurrence of Ca/2+ sparks under either ligand- or voltage- activation in order to determine the effects of cytosolic [Mg/2+] and [Ca/2+], which are major physiological modulators of both voltage- and ligand-gated events. By determining the concentration dependence and interaction of these two divalent cations on event frequencies and patterns we will develop and test gating schemes for both types of activation and determine whether the same mechanistic steps underlie both ligand- and voltage-gated events. Determining the frequency of sparks under various conditions in these studies will provide the first characterization of the opening rates of SR channels in functioning muscle fibers. We will also use a diffusion and binding model, including diffusion of Ca/2+, CaDye and the sarcomeric distribution of Ca/2+ binding sites, to stimulate sparks and to estimate the amount of Ca/2+ related in a spark. Finally, we will determine whether a spark is generated by the opening of more than one SR Ca/2+ channel. These studies will provide deeper insight into the cellular and molecular mechanisms underlying regulation of Ca/2+ release in skeletal muscle in various physiological states as well as under possible pathological conditions.